TY - JOUR A1 - Oprzeska-Zingrebe, Ewa Anna A1 - Meyer, Susann A1 - Roloff, Alexander A1 - Kunte, Hans-Jörg A1 - Smiatek, Jens T1 - Influence of compatible solute ectoine on distinct DNA structures BT - thermodynamic insights into molecular binding mechanisms and destabilization effects JF - Physical chemistry, chemical physics : a journal of European Chemical Societies N2 - In nature, the cellular environment of DNA includes not only water and ions, but also other components and co-solutes, which can exert both stabilizing and destabilizing effects on particular oligonucleotide conformations. Among them, ectoine, known as an important osmoprotectant organic co-solute in a broad range of pharmaceutical products, turns out to be of particular relevance. In this article, we study the influence of ectoine on a short single-stranded DNA fragment and on double-stranded helical B-DNA in aqueous solution by means of atomistic molecular dynamics (MD) simulations in combination with molecular theories of solution. Our results demonstrate a conformation-dependent binding behavior of ectoine, which favors the unfolded state of DNA by a combination of electrostatic and dispersion interactions. In conjunction with the Kirkwood-Buff theory, we introduce a simple framework to compute the influence of ectoine on the DNA melting temperature. Our findings reveal a significant linear decrease of the melting temperature with increasing ectoine concentration, which is found to be in qualitative agreement with results from denaturation experiments. The outcomes of our computer simulations provide a detailed mechanistic rationale for the surprising destabilizing influence of ectoine on distinct DNA structures. Y1 - 2018 U6 - https://doi.org/10.1039/c8cp03543a SN - 1463-9076 SN - 1463-9084 VL - 20 IS - 40 SP - 25861 EP - 25874 PB - Royal Society of Chemistry CY - Cambridge ER - TY - JOUR A1 - Hahn, Marc Benjamin A1 - Meyer, Susann A1 - Schröter, Maria-Astrid A1 - Seitz, Harald A1 - Kunte, Hans-Jörg A1 - Solomun, Tihomir A1 - Sturm, Heinz T1 - Direct electron irradiation of DNA in a fully aqueous environment BT - Damage determination in combination with Monte Carlo simulations JF - Physical chemistry, chemical physics : PCCP ; a journal of European chemical societies N2 - We report on a study in which plasmid DNA in water was irradiated with 30 keV electrons generated by a scanning electron microscope and passed through a 100 nm thick Si3N4 membrane. The corresponding Monte Carlo simulations suggest that the kinetic energy spectrum of the electrons throughout the water is dominated by low energy electrons (<100 eV). The DNA radiation damage, single-strand breaks (SSBs) and double-strand breaks (DSBs), was determined by gel electrophoresis. The median lethal dose of D-1/2 = 1.7 +/- 0.3 Gy was found to be much smaller as compared to partially or fully hydrated DNA irradiated under vacuum conditions. The ratio of the DSBs to SSBs was found to be 1 : 12 as compared to 1 : 88 found for hydrated DNA. Our method enables quantitative measurements of radiation damage to biomolecules (DNA, proteins) in solutions under varying conditions (pH, salinity, co-solutes) for an electron energy range which is difficult to probe by standard methods. Y1 - 2016 U6 - https://doi.org/10.1039/c6cp07707b SN - 1463-9076 SN - 1463-9084 VL - 19 IS - 3 SP - 1798 EP - 1805 PB - RSC Publ. CY - Cambridge ER - TY - JOUR A1 - Hahn, Marc Benjamin A1 - Solomun, Tihomir A1 - Wellhausen, Robert A1 - Hermann, Sabrina A1 - Seitz, Harald A1 - Meyer, Susann A1 - Kunte, Hans-Jörg A1 - Zeman, Johannes A1 - Uhlig, Frank A1 - Smiatek, Jens A1 - Sturm, Heinz T1 - Influence of the Compatible Solute Ectoine on the Local Water Structure: Implications for the Binding of the Protein G5P to DNA JF - The journal of physical chemistry : B, Condensed matter, materials, surfaces, interfaces & biophysical chemistry N2 - Microorganisms accumulate molar concentrations of compatible solutes like ectoine to prevent proteins from denaturation. Direct structural or spectroscopic information on the mechanism and about the hydration shell around ectoine are scarce. We combined surface plasmon resonance (SPR), confocal Raman spectroscopy, molecular dynamics simulations, and density functional theory (DFT) calculations to study the local hydration shell around ectoine and its influence on the binding of a gene-S-protein (G5P) to a single-stranded DNA (dT(25)). Due to the very high hygroscopicity of ectoine, it was possible to analyze the highly stable hydration shell by confocal Raman spectroscopy. Corresponding molecular dynamics simulation results revealed a significant change of the water dielectric constant in the presence of a high molar ectoine concentration as compared to pure water. The SPR data showed that the amount of protein bound to DNA decreases in the presence of ectoine, and hence, the protein-DNA dissociation constant increases in a concentration-dependent manner. Concomitantly, the Raman spectra in terms of the amide I region revealed large changes in the protein secondary structure. Our results indicate that ectoine strongly affects the molecular recognition between the protein and the oligonudeotide, which has important consequences for osmotic regulation mechanisms. Y1 - 2015 U6 - https://doi.org/10.1021/acs.jpcb.5b09506 SN - 1520-6106 VL - 119 IS - 49 SP - 15212 EP - 15220 PB - American Chemical Society CY - Washington ER - TY - JOUR A1 - Kuhne, Maren A1 - Dippong, Martin A1 - Flemig, Sabine A1 - Hoffmann, Katrin A1 - Petsch, Kristin A1 - Schenk, Jörg A. A1 - Kunte, Hans-Jörg A1 - Schneider, Rudolf J. T1 - Comparative characterization of mAb producing hapten-specific hybridoma cells by flow cytometric analysis and ELISA JF - Journal of immunological methods N2 - A novel method that optimizes the screening for antibody-secreting hapten-specific hybridoma cells by using flow cytometry is described. Cell clones specific for five different haptens were analyzed. We selectively double stained and analyzed fixed hybridoma cells with fluorophore-labeled haptens to demonstrate the target-selectivity, and with a fluorophore-labeled anti-mouse IgG antibody to characterize the level of surface expression of membrane-bound IgGs. ELISA measurements with the supernatants of the individual hybridoma clones revealed that antibodies from those cells, which showed the highest fluorescence intensities in the flow cytometric analysis, also displayed the highest affinities for the target antigens. The fluorescence intensity of antibody-producing cells corresponded well with the produced antibodies' affinities toward their respective antigens. Immunohistochemical staining verified the successful double labeling of the cells. Our method makes it possible to perform a high-throughput screening for hybridoma cells, which have both an adequate IgG production rate and a high target affinity. (C) 2014 Elsevier B.V. All rights reserved. KW - Immunization KW - Hapten KW - Monoclonal antibodies KW - Hybridoma KW - Flow cytometry KW - ELISA Y1 - 2014 U6 - https://doi.org/10.1016/j.jim.2014.07.004 SN - 0022-1759 SN - 1872-7905 VL - 413 SP - 45 EP - 56 PB - Elsevier CY - Amsterdam ER -